Induction of Human Embryonic Stem Cells into neuronal differentiation by increasing cyclic Adenosine Mono Phosphate

Introduction: To evaluate the cAMP -mediated IBMX (3-IsoButyle -1-Methyl Xanthin) and db-cAMP (dibutyryl cAMP) effects on differentiation of human Embryonic Stem Cells (hESCs) into nerve cells were the objectives of this study. Methods: We have used Royan H1 hESC- derived embryoid bodies with four treatment groups: six days treatment with IBMX (5×10 -4M) and db-cAMP (10 -9M) (referred to as cAMP), retinoic acid (RA, 10 -6M), IBMX + db-cAMP + RA, and control (no treatment). Immunocytochemistry was carried out for neural specific antibodies including β-Tubulin III, Microtubule Associated Protein 2 (MAP-2), Neurofilament Protein-Heavy chain (NF-H), Glial Fibrilary Acidic Protein (GFAP) and Synaptophysin as well as morphological studies. Semiquantitative RT-PCR was also used to evaluate gene expression involved in neurogenesis. Results: In the 4+6+4 days the neuronal process were apparently observed. Immunocytochemical studies using nerve specific antibodies for proteins such as β- Tubulin III, MAP-2, NF-H, GFAP and Synaptophysin showed the presence of these neuronal and astrocyte markers in differentiated cells by cAMP. Evaluation of expression of genes involved in neurogenesis showed that Hash1, Synaptophysin, β-Adrenergic Receptor and Acetylcholine Receptor- which were silent in embryoid bodies - switched on after treatment with cAMP and/or RA. Relative expression of nerve specific genes showed a significant enhancement in expression of Synaptophysin, NFM and β-Adrenergic Receptor during differentiation, which, with the enhancement in cAMP treated groups were more than those treated with RA and control (p < 0.05). Conclusion: In conclusion, this study showed that cAMP could be a neurogenic agent for human embryonic stem cells differentiation.